The DNA sequence and genetic organization of a Neurospora mitochondrial plasmid suggest a relationship to introns and mobile elements

Nargang, Frank E.; Bell, John B.; Stohl, Lori L.; Lambowitz, Alan M.

doi:10.1016/0092-8674(84)90499-9

Cited by 134 publications

(56 citation statements)

References 52 publications

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“…The plasmids in strains Mauriceville and Varkud contain one long open reading frame, consisting of short blocks of amino acids that are characteristic of reverse transcriptase (Nargang et al, 1984;Akins et al. 1988).…”

Section: Neurosporamentioning

confidence: 99%

tRNA‐like structures

Mans

Pleij

Bosch

1991

European Journal of Biochemistry

122

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Section: Neurosporamentioning

confidence: 99%

tRNA‐like structures

Mans

Pleij

Bosch

1991

European Journal of Biochemistry

122

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“…Prediction of secondary structure and calculation of AG values were based on a computer program developed by Zuker and Stiegler [22], adapted for use on an IBM AT. Other analyses utilized a consolidation [23] of the programs of Staden.…”

Section: N a Techniquesmentioning

confidence: 99%

Cloning, nucleotide sequence and expression of the pyr BI operon of Salmonella typhimurium LT2

1987

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The pyrB-pyrI region of the Salmonella typhimurium LT2 chromosome has been cloned and sequenced. The two genes were found to constitute an operon, with pyrl being the distal gene and separated from pyrB by a 15-bp intercistronic region. Sequence analysis revealed the presence of two potential promoters; transcription initiated from the promoter proximal to pyrB would produce a transcript which could direct the synthesis of a 33-aminoacid leader peptide. The leader sequence possesses the requisite features of a rho-independent transcriptional terminator (attenuator) which is positioned 22 bp upstream from the pyrB structural gene. A regulatory mutation imparting a 30-fold elevated expression of pyrBI was identified as a two-base-pair deletion in the track of A . T base pairs located on the 3' side of the region of dyad symmetry of the attenuator. The leader sequence also has an additional region of dyad symmetry (putative transcriptional pause site) located 33 nucleotides upstream from the start of the proposed attenuator. The intervening sequence between the putative pause site and the indicated attenuator is characterized by encoding a high content of uracil residues in the transcript. Construction and analysis of transcriptional and translational fusions provided evidence that (a) the leader region has the necessary features to mediate polypeptide synthesis in vivo, (b) the removal of the region corresponding to the pause site and attenuator results in constitutive expression and (c) the more distant potential promoter does not appear to facilitate significant transcriptional activity. Strong homology exists with the pyrBI operon from E . s c~~r i c~i a coli K-12 but notable differences are observed.The pyrimidine biosynthetic enzyme, aspartate transcarbamoylase from Escherichia coli and Sulnionella typhimurium is composed of six catalytic (c) and six regulatory (r) polypeptides, which are organized as two trimeric catalytic (C) subunits and three dimeric regulatory (R) subunits in the native enzyme (reviewed in [l]). The structural genes for the c and r polypeptides have been designated pyrB and pyrl, respectively, and for E. coli the two genes have been shown to comprise an operon, with pyrB being promoter-proximal Previous studies have shown that expression of pyrBI is regulated over a several-hundredfold range [3 -51 and that regulation of expression occurs at the level of transcription [6]. Sequencing studies on the promoter-regulatory region of pyrBI from E. coli led to the identification of a putative rhoindependent terminator (attenuator) located approximately 20 bp upstream of the pyrB gene [7, 81. Also identified was a transcriptional pause site positioned roughly 20 bp before the start of the attenuator region [8]. The region at the pause site encodes a uridine-rich sequence in the transcript. Transcription initiating from a promoter about 160 bp upstream of pyrB, and proceeding without termination at the attenuator, will produce a transcript which can encode a 44-amino-acid peptide, with the termin...

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“…First, the doublestranded plasmid DNA is transcribed by the host Neurospora sp. mitochondrial (mt) RNA polymerase to yield full-length linear RNAs (20,31 plasmid's replication (23). The major in vitro transcription start site is -260 bp upstream of the 5' end of the plasmid transcript, suggesting that monomer-length transcripts are generated by transcription around the plasmid coupled with site-specific cleavage at the position between the 5' and 3' ends of the RNA (20).…”

mentioning

confidence: 99%

A mitochondrial retroplasmid integrates into mitochondrial DNA by a novel mechanism involving the synthesis of a hybrid cDNA and homologous recombination.

et al. 1994

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The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, small circular DNAs that propagate via an RNA intermediate and reverse transcription. Although the plasmids ordinarily replicate autonomously, they can also integrate into mitochondrial DNA (mtDNA), yielding defective mtDNAs that in some cases cause senescence. To investigate the integration mechanism, we analyzed four cases in which the Varkud plasmid integrated into the mitochondrial small rRNA gene, three in wild-type subcultures and one in a senescent mutant. Our analysis suggests that the integrations occurred by the plasmid reverse transcriptase template switching between the plasmid transcript and internal sequences in the mitochondrial small rRNA to yield hybrid cDNAs that circularized and recombined homologously with the mtDNA. The integrated plasmid sequences are transcribed, presumably from the mitochondrial small rRNA promoters, resulting in hybrid RNAs containing the 5' segment of the mitochondrial small rRNA linked head-to-tail to the full-length plasmid transcript. Analysis of additional senescent mutants revealed three cases in which the plasmid used the same mechanism to integrate at other locations in the mtDNA. In these cases, circular variant plasmids that had incorporated a mitochondrial tRNA or tRNA-like sequence by template switching integrated by homologous recombination at the site of the corresponding tRNA or tRNA-like sequence in mtDNA. This simple integration mechanism involving template switching to generate a hybrid cDNA that integrates homologously could have been used by primitive retroelements prior to the acquisition of a specialized integration machinery.The Mauriceville plasmid and closely related Varkud plasmid, found in the mitochondria of some Neurospora strains, are novel retroelements that may be related to retroviral ancestors (23,38). The plasmids exist predominantly as small circular DNAs and concatemers but appear to replicate via an RNA intermediate and reverse transcription. As shown in Fig. 1, Mauriceville and Varkud plasmid monomers are circular double-stranded DNAs of 3.6 and 3.7 kb, respectively, and contain a single long open reading frame (ORF) of 710 amino acids (2, 9, 31). This ORF encodes an 81-kDa reverse transcriptase (RT) protein that is highly conserved between the two plasmids and presumably functions in their replication (24). The plasmid RT contains conserved amino acid sequence blocks 1 to 7, found in most RTs, as well as an additional sequence block, 2a, characteristic of the non-long terminal repeat (non-LTR) retroelements (14, 39). Among the non-LTR retroelements, the plasmid RT is most closely related to those encoded by bacterial retrons and group II introns (14, 39). As expected for a primitive retroelement, the plasmid lacks motifs characteristic of gag, RNase H, protease, integrase, or Zn2+ fingerlike domains, which are associated with the RTs of other retroelements (24,26,27,37).Biochemical experiments have provided insight into the replication mechan...

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The DNA sequence and genetic organization of a Neurospora mitochondrial plasmid suggest a relationship to introns and mobile elements

Cited by 134 publications

References 52 publications

tRNA‐like structures

tRNA‐like structures

Cloning, nucleotide sequence and expression of the pyr BI operon of Salmonella typhimurium LT2

A mitochondrial retroplasmid integrates into mitochondrial DNA by a novel mechanism involving the synthesis of a hybrid cDNA and homologous recombination.

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